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Cell Death and Differentiation (2006) 13, 84–95
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AtBAG6, a novel calmodulin-binding protein, induces
programmed cell death in yeast and plants
CH Kang1, WY Jung1, YH Kang1, JY Kim1, DG Kim1, JC Jeong1,
DW Baek1, JB Jin1, JY Lee1, MO Kim1, WS Chung1, T Mengiste2,
H Koiwa3, SS Kwak4, JD Bahk1, SY Lee1, JS Nam5, DJ Yun*,1 and
MJ Cho1
1
Division of Applied Life Science (BK21 program) and Environmental
Biotechnology National Core Research Center, Graduate School of
Gyeongsang National University, Jinju 660-701, Korea
2
Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN 47907-2054, USA
3
Department of Horticultural Sciences, Texas A&M University, College Station,
TX 77843-2133, USA
4
Laboratory of Environmental Biotechnology, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), Yusong, Daejeon 305-806, Korea
5
Faculty of Plant Biotechnology, Dong-A University, Busan 604-714, Korea
* Corresponding author: DJ Yun, Division of Applied Life Science (BK21
program) and Environmental Biotechnology National Core Research Center,
Gyeongsang National University, #900 Gajwa Dong, Jinju 660-701, Korea.
Tel: þ 82-55-751-6256; Fax: þ 82-55-759-9363;
E-mail: [email protected]
Received 10.9.04; revised 30.5.05; accepted 30.5.05; published online 08.7.05
Edited by B Zhivotovsky
Abstract
Calmodulin (CaM) influences many cellular processes by
interacting with various proteins. Here, we isolated AtBAG6,
an Arabidopsis CaM-binding protein that contains a central
BCL-2-associated athanogene (BAG) domain. In yeast and
plants, overexpression of AtBAG6 induced cell death
phenotypes consistent with programmed cell death (PCD).
Recombinant AtBAG6 had higher affinity for CaM in the
absence of free Ca2 þ than in its presence. An IQ motif
(IQXXXRGXXXR, where X denotes any amino-acid) was
required for Ca2 þ -independent CaM complex formation and
single amino-acid changes within this motif abrogated both
AtBAG6-activated CaM-binding and cell death in yeast and
plants. A 134-amino-acid stretch, encompassing both the IQ
motif and BAG domain, was sufficient to induce cell death.
Agents generating oxygen radicals, which are known to be
involved in plant PCD, specifically induced the AtBAG6
transcript. Collectively, these results suggest that AtBAG6 is
a stress-upregulated CaM-binding protein involved in plant
PCD.
Cell Death and Differentiation (2006) 13, 84–95.
doi:10.1038/sj.cdd.4401712; published online 8 July 2005
Keywords: calcium; calmodulin; CaM-binding protein; IQ motif;
BAG domain; stress; Arabidopsis
Abbreviations: CaM, calmodulin; CaMBPs, CaM-binding proteins; BAG, BCL-2-associated athanogene; AtBAG, Arabidopsis
thaliana BAG; CDD, cell death domain; GST, glutathione
S-transferase; HRP, horse radish peroxidase; PCD, programmed
cell death; ROS, reactive oxygen species
Introduction
In both plants and animals, the cytosolic free calcium level
([Ca2 þ ]cyt) is normally maintained at submicromolar levels,
but can be transiently elevated with complex forms of
amplitude, frequency and duration by specific external
stimuli.1,2 Transiently elevated [Ca2 þ ]cyt in the cell leads to
binding of Ca2 þ to intracellular regulatory proteins, initiating a
wide variety of cellular processes.3 Calmodulin (CaM) is a
ubiquitous Ca2 þ receptor protein that regulates the activities
and functions of a wide range of CaM-binding proteins
(CaMBPs), including metabolic enzymes, transcription factors, ion channels, protein kinases/phosphatases, and structural proteins.4,5
The overall shape of CaM is a dumbbell, with the N- and Cterminal globular domains separated by a flexible central
helix. Ca2 þ -binding to CaM induces conformational changes
that facilitate binding of Ca2 þ /CaM-dependent target proteins
and subsequent modulation of their biological activities.6
Although considerable research has focused on the Ca2 þ bound form of CaM, it is known that the Ca2 þ -free state of
CaM, apocalmodulin (ApoCaM), also binds to other (or
overlapping) proteins and signals cellular responses.7,8
ApoCaM differs from Ca2 þ -bound CaM in its three-dimensional structure, and it also binds target proteins differently,
utilizing binding motifs such as the IQ motif (IQXXXRGXXXR)
and noncontiguous binding sites.9,10 Therefore, CaM may
cycle between Ca2 þ -free and Ca2 þ -bound states, and bind
different proteins in each state to trigger specific cellular
functions.10
In plant cells, Ca2 þ -signaling is implicated in diverse
processes, including thigmomorphogenesis, light signal
transduction, gibberellic acid signaling, pathogen-challenged
defense signaling, and salt stress signaling.11,12 Since CaM is
known to transduce Ca2 þ -signals by modulating the activity of
numerous and diverse CaMBPs, thereby generating physiological responses to various stimuli, many CaM and CaMBPs
have been isolated with a view to furthering our understanding
of Ca2 þ -mediated plant signaling pathways.13–15 However,
many aspects of plant CaM and CaMBPs signaling, as well as
their functional significance, remain unclear.
BAG (BCL2-associated athanogene) family proteins were
originally identified in mammals due to their ability to associate
with the antiapoptotic protein, BCL2, and promote cell
survival.16 Further studies revealed that BAG family genes
exist in a number of eukaryotes whose members share at
least one copy of a 50 amino-acid conserved domain (BAG
domain) that interacts with heat shock protein 70 (HSP70/
HSC70).16–18 BAG proteins may serve as bridging molecules
that recruit HSP70/HSC70 to a specific target protein. As a
consequence, this family of cochaperones functionally
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
85
regulates diverse cellular pathways, such as programmed cell
death (PCD) and stress responses.19 While information is
available on the biological roles of BAG proteins in mammals,
no plant homologs of BAG family members have been
functionally characterized to date.
To determine the biological role(s) of CaM in plants, we
have previously isolated a number of CaMBP(s).20–22 Here,
we report the isolation and characterization of an Arabidopsis
gene encoding a BAG protein homolog (AtBAG6). Our data
indicate that AtBAG6 expression in yeast and plant cells
induces cell death. Furthermore, the IQ motif within the
AtBAB6 sequence is required for Ca2 þ -independent complex
formation with CaM and AtBAG6-mediated cell death in yeast
and plant. This is the first documented evidence that a BAG
domain protein is involved in Ca2 þ /CaM-mediated signaling
in eukaryotes.
Results
Cloning AtBAG6 encoding a CaM-binding protein
To unequivocally establish the role of CaM-mediated signaling in plants, many CaMBPs that may link Ca2 þ /CaM signals
to specific responses have been cloned.13,14,23 In this study,
we screened an Arabidopsis cDNA expression library
constructed from heat-treated plant seedlings using AtCaM2::HRP as a probe.20,22 In all, 11 positive clones were
obtained from a total of 5 105 recombinant phages. Based
on the restriction patterns of plasmid inserts, the genes coding
for CaMBPs were classified into seven different loci. Since the
protein encoding CaMBP1 conferred the highest CaM-binding
activity (data not shown), this gene was selected for further
study. Proteins encoded by other CaMBPs are not structurally
related to CaMBP1, and will be described elsewhere.
Sequence determination of CaMBP1 and comparison to
sequences within GenBankTM revealed that the nucleotide
sequence of the 2105 bp cDNA clone contains a partial coding
region (encoding 674 amino acids) and a full 30 -untranslated
region. The 50 -truncated region of the cDNA was recovered by
RACE-PCR, and sequenced. The 3170 nucleotide full-length
cDNA contained an open reading frame encoding a putative
117 kDa protein consisting of 1043 amino-acid residues
identical to AtBAG6 (accession no. At2g46240).24 Accordingly, CaMBP1 was redesignated AtBAG6 (Figure 1). In
mammals, BAG domain proteins interact with other proteins to
modulate cellular processes. To identify the plant proteins
Figure 1 Deduced amino-acid sequence of AtBAG6 and comparison of its putative CaMBD (IQ motif) to ones of other CaM-binding proteins. (a) Deduced amino-acid
sequence of AtBAG6. The predicted CaMBD (light gray), BAG domain (underlined) and glutamic acid-rich regions (dark grey) are indicated. (b) Schematic representation
of AtBAG6 and alignment of protein sequences from AtBAG6 with CaMBDs from AtBAG5 (A. thaliana BAG domain containing protein; NP_172670), AtEICBP (A.
thaliana EICBP protein; AC007168), LeER66 (Lycopersicon esculentum ER66 protein; AAD46410), MYA1 (A. thaliana myosin MYA1; S46444), NtcNMP-RIC (Nicotiana
tabacum CaMB-channel protein; AAB53255), HaSF16 PSP (Helianthus annuus SF16 protein; CAA52782), and ATM2 (A. thaliana myosin MYA1; S46444). Amino-acid
residues identical to the consensus CaMBD sequences are depicted in black
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associated with AtBAG6, we employed the yeast two-hybrid
screening system using full-length AtBAG6 as bait. We
identified 33 yeast colonies that expressed both reporter
genes (Ade þ /lacZ þ ) in an AtBAG6-dependent manner.
Sequence analyses revealed that all the AtBAG6-associated
proteins are AtCaM isoforms, specifically, five AtCaM1s
(accession no. At5g37780), 14 AtCaM2s (accession no.
At2g41110), seven AtCaM3s (accession no. At3g56800),
two AtCaM4s (accession no. At1g66410), three AtCaM6s
(accession no. At5g21274) and two AtCaM7s (accession no.
At3g43810). These results indicate that AtBAG6 mainly
interacts with AtCaM isoforms in vivo.
Mapping of CaMBD within AtBAG6
Comparative sequence analysis of AtBAG6 with other known
proteins revealed the presence of a putative CaM-binding IQ
motif25–28 and a BAG domain17–19 in the middle of the
sequence (Figure 1). A glutamic acid-rich region was
identified near the carboxyl terminus of the protein sequence
on a database using the ScanProsite program (http://
www.expasy.org/cgi-bin/scanprosite).29 To determine the
CaM-binding regions in AtBAG6, we performed the
CaM::HRP overlay assay with Escherichia coli-expressed
recombinant AtBAG6 proteins. To this end, we generated
GST-fused constructs containing full-length cDNA (designated D0) and five serial deletion derivatives of AtBAG6 (D1,
D2, D3, D4, and CDD) (Figure 2a). Recombinant proteins
were produced in E. coli, purified, and verified by anti-GST
polyclonal antibodies. The molecular weight (MW) of recombinant AtBAG6 was much higher than the calculated size (the
expected MW of D0 is about 143 kDa) (Figure 2b). This
difference may result from the anomalous electrophoretic
mobility caused by the large number of Glu residues within the
AtBABG6 sequence, which reduce SDS binding.30,31 Four
recombinant proteins (D0, D2, D3, and CDD) containing the
putative CaMBD interacted with AtCaM2::HRP, whereas GST
only (data not shown) and GST fusion proteins lacking the
predicted IQ motif (D1 and D4) did not interact
with AtCaM2::HRP (Figure 2b). Importantly, CaM bound
AtBAG6 in the absence, but not the presence of Ca2 þ . Similar
results were obtained when 6-His-tagged AtBAG6 recombinant proteins were used to perform this assay (data not
shown).
Crucial residues of the CaM-binding motif
Comparative analysis of the CaM-binding regions of many
CaMBP proteins has led to the identification of multiple
sequence motifs required for CaM complex formation.,26 We
identified a putative IQ motif, a structural characteristic of
CaMBDs that has been identified previously, between Ala572
and Lys591 of AtBAG6 (Figure 1). To further determine
whether the IQ motif in the region is required for CaM-binding,
we separately substituted Ile575 in the CDD (described in
Figure 1a) with Val, Ser, and Asn (designated CDDI575V,
CDDI575S and CDDI575N, respectively) (Figure 3a). Recombinant proteins were produced in E. coli and analyzed for CaMbinding activity using the CaM overlay assay. CaM bound to
the wild-type CDD and CDDI575V but not to CDDI575S or
Cell Death and Differentiation
Figure 2 Identification of the CaM-binding domain of AtBAG6. (a) Schematic
representation of AtBAG6 (D0) and serial fragment constructs (D1–D4, and
CDD). The IQ motif and BAG domain are depicted by a circle and a box,
respectively. Amino-acid positions of each serial fragment are indicated. D0–D4,
and CDD represent six GST fusion constructs containing the indicated fragments
of AtBAG6. CaM-binding ability is indicated as Yes (CaM-binding) or No (no
CaM-binding). (b) CaM-binding analysis. Six GST fusion proteins of serial
fragment mutants (D0–D4, and CDD) of AtBAG6 were produced in E. coli. The
recombinant proteins were analyzed by Western blotting with an anti-GST
antibody (left panel). The CaM::HRP overlay assay was performed in the
absence (5 mM EGTA, middle panel) or presence (1 mM CaCl2, right panel) of
Ca2 þ
CDDI575N mutant proteins (Figure 3a). Similar results were
obtained when we performed the yeast two-hybrid assay with
full-length AtBAG6 and AtCaM2 (Figure 3b). Taken together,
these results indicate that Ile575 in AtBAG6 is crucial for
interactions and specific complex formation with AtCaMs.
Induction of cell death by AtBAG6 in yeast
The BAG domain was originally identified in human cells,
based on its ability to bind to BCL2 (an antiapoptotic protein)
and promote cell survival.16,32 Accordingly, we investigated
whether the biological function of AtBAG6 is associated with
cell survival by expressing the protein in a unicellular yeast
system. A cDNA encoding full-length AtBAG6 was subcloned
into the episomal pYES2 vector under control of the GAL1
promoter, which allows for the conditional expression of this
protein when cells are grown in galactose-containing medium.
Unexpectedly, we found that expression of AtBAG6 in yeast
cells induced cell death (Figure 4a).
More than eight genes have been identified in Arabidopsis
that encode proteins with a BAG domain.24 To investigate
whether other members of this family are also involved in
yeast cell death, two AtBAG6 paralogues, AtBAG1
(At5g52060) and AtBAG8 (At3g29310), were transformed
into yeast cells. The amino acid similarities between the BAG
domain of AtBAG6 and those of AtBAG1 and AtBAG8 are 58,
and 52%, respectively. Expression of AtBAG6 dramatically
A CaM-binding BAG domain protein in Arabidopsis
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Figure 3 Characterization of the IQ motif of AtBAG6. (a) CaM-binding to the IQ
motif of CDD in vitro. CDD mutants that contain single amino-acid substitutions
were fused to the C-terminus of GST, and expressed in E. coli. The recombinant
proteins were analyzed by Western blotting with an anti-GST antibody (left
panel). The CaM::HRP overlay assay (described in Materials and Methods) was
performed in the absence (5 mM EGTA, middle panel) or presence (1 mM CaCl2,
right panel) of Ca2 þ . CDDI575V, CDDI575S and CDDI575N represent replacement
of Ile575 in the IQ motif with Val, Ser, and Asn, respectively. (b) Interactions
between AtBAG6 and AtCaM2 in yeast. The indicated combinations of bait
(pGBT9) and prey (pGAD424) constructs were transformed into the yeast
reporter strain, PJG69-4. Transformants were examined for growth in the
absence of Trp, Leu (SD-WL) or Trp, Leu, Ade (SD-WLA) and for the bgalactosidase assay. (1) interactions between tumor suppressor p53 (pTD1-1)
and simian virus 40 large T-antigen (pVA3-1); (2) interactions between wild-type
AtBAG6 (BD::AtBAG6) and AtCaM2 (AD::AtCaM2); (3) interactions between
AtBAG6I575V (BD::AtBAG6I575V) and AtCaM2 (AD::AtCaM2); (4) interactions
between AtBAG6I575S (BD::AtBAG6I575S) and AtCaM2 (AD::AtCaM2); (5)
interactions between AtBAG6I575N (BD::AtBAG6I575N) and AtCaM2 (AD::AtCaM2); (6) BD vector (pGBT9) and AD::AtCaM2; (7) BD::AtBAG6 and AD vector
(pGAD424); (8) BD::AtBAG6I575V and AD vector (pGAD424); (9) BD::AtBAG6I575S and AD vector (pGAD424); 10, BD::AtBAG6I575N and AD vector
(pGAD424)
induced cell death; however, transformants expressing
AtBAG1 or AtBAG8 did not exhibit cell death (Figure 4a).
This suggests that, of the Arabidopsis BAG proteins, AtBAG6
is a specific cell death inducer.
The cell death phenotype induced by AtBAG6 was further
confirmed by trypan blue exclusion and 40 , 60 -diamino-2phenylindole dihydrochloride (DAPI) staining (Figure 4b and
data not shown). DAPI staining revealed that the majority of
cells not expressing AtBAG6 had a normal and single roundshaped nucleus, whereas an abundance of abnormally
shaped and fragmented nuclei (approximately 50%) was
observed in the cells expressing AtBAG6 (Figure 4b).
Expression of AtBAG6 also resulted in exposure of phosphatidylserine on the surface of the cytoplasmic membrane, as
revealed by FITC-annexin V staining (Figure 4c), as well as
the occurrence of DNA strand breaks, demonstrated by
TUNEL assay (Figure 4b).33,34 Furthermore, alterations
associated with cell death induced by AtBAG6 included
abnormal morphology, cell shrinkage, increased vacuolation
Figure 4 AtBAG6 induces cell death in yeast. (a) Functional analysis of
Arabidopsis BAG family proteins in yeast cells. Yeast cells transformed with
plasmid pYES2 (control) containing BAX (Moon et al.,85), AtBAG6, AtBAG1, or
AtBAG8 were cultured in glucose-based medium to an OD600 of B1.0 at 301C
for 12 h. Equal numbers of cells were spotted on minimal SD medium plates in
the presence of glucose or galactose, as described in Materials and Methods.
Photographs were taken after culturing at 301C for 2 days. (b, c, and d) AtBAG6induced programmed cell death in yeast. Yeast cells transformed with plasmid
pYES2 (control) containing AtBAG6 grown on either glucose or galactose were
stained with DAPI-TUNEL (for detection of cell death and DNA strand breaks),
Annexin V-PI (for detection of exposed phophatidylserine), or dihydrorhodamine123 (Rh123, for detection of reactive oxygen generation), respectively.
Experimental conditions were described in Materials and Methods. Scale bar,
5 mm
without membrane rupture, and loss of plasma membrane
integrity (data not shown). All these morphological characteristics are hallmarks of apoptosis.34–36
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Reactive oxygen species (ROS) have been implicated as
effectors of PCD in animal and yeast cells.37,38 Accordingly,
the role of ROS in AtBAG6-induced cell death was examined.
Production of ROS was monitored using dihydrorhodamine123. Upon oxidation by ROS, nonfluorescent dihydrorhodamine123 changes into the fluorescent chromophore,
rhodamine123.39 As shown in Figure 4d, yeast cells expressing AtBAG6 exhibited strong fluorescence when incubated
with dihydrorhodamine123, whereas control cells grown in
glucose-containing medium exhibited no significant fluorescence, suggesting that the generation of ROS may mediate
AtBAG6-induced cell death in yeast.
Requirement of the IQ motif and BAG domain for
AtBAG6-mediated cell death
To identify the critical domain in AtBAG6 responsible for
inducing cell death, deletion mutant clones of AtBAG6
(depicted in Figure 2a and Supplementary Figure 1) were
subcloned into the pYES2 vector and cell death phenotypes
were investigated (Figure 5 and Supplementary Figure 1).
Colonies formed by yeast cells harboring all of the constructs
on glucose-based medium were detected with approximately
the same efficiency as control transformants (Figure 5, left
panel). However, transformants harboring both the IQ motif
and BAG domain (D0, D3, CDD) showed greatly reduced
colony formation on galactose medium (Figure 5, right panel).
To further investigate the significance of CaM–AtBAG6
interactions in AtBAG6- mediated cell death, we investigated
the cell death phenotype of AtBAG6 containing a single
amino-acid substitution in the IQ motif (described in Figure 3).
Transformants harboring full-length AtBAG6 or AtBAG6I575V
(Ile575 substituted with Val) resulted in a dramatic decrease in
colony formation and cell viability on galactose medium.
However, those containing AtBAG6I575S or AtBAG6I575N
(Ile575 substituted with Ser or Asn, respectively) displayed
similar colony-forming efficiency in glucose- or galactose-
Figure 5 Identification of the cell death domain in AtBAG6. The specified
constructs depicted in Figure 2 were transformed into the yeast strain, W303a.
Equal numbers of cells were spotted on minimal SD medium plates in the
presence of glucose or galactose, as described in Materials and Methods.
Photographs were taken after culturing at 301C for 2 days. Inducibility of cell
death is indicated as Yes (inducible) or No (not inducible)
Cell Death and Differentiation
based medium (Figure 6a). Similar results were obtained
when the CDD domain was tested with these mutations
(Figure 6b and data not shown). Taken together, these results
provide strong evidence that the CaM-binding domain is
required for AtBAG6-mediated cell death in yeast.
AtBAG6 does not bind AtHSC70, an Arabidopsis
heat shock protein
Since BAG family proteins in animals are known to interact
with and regulate the activity of HSP70/HSC70 (heat shock
proteins of relative molecular mass 70 kDa) family molecular
chaperones, we investigated whether AtBAG6 interacted with
AtHSC70 (accession no. X74604) a plant homolog of
animal HSP70/HSC70. GST fusion proteins of AtBAG6, CDD
(a fragment from Gln560 to Pro693 of AtBAG6), and two
AtBAG6 paralogues, AtBAG3 (At5g07220) and AtBAG5
(At1g12060), were produced in E. coli and analyzed for their
ability to interact with 6-His-tagged AtHSC70 by far-Western
blot analysis.16,17 AtBAG3 and AtBAG5 bound to the
AtHSC70, whereas AtBAG6 and CDD did not (Figure 7 and
data not shown).
Figure 6 CaM-binding IQ motif is required for AtBAG6-induced cell death. (a)
pYES2 vector (control) containing full-length AtBAG6, or AtBAG6 mutants that
contain single amino-acid substitutions were transformed into a yeast strain,
W303a. Equal numbers of cells were spotted on minimal SD medium plates in the
presence of glucose or galactose, as described in Materials and Methods.
Photographs were taken after culturing at 301C for 2 days. AtBAG6I575V,
AtBAG6I575S and AtBAG6I575N represent replacement of Ile575 in the AtBAG6
with Val, Ser, and Asn, respectively. (b) pYES2 vector (control) containing a CDD
(depicted in Figure 2), or a CDD mutant (CDDI575S substitution of Ile575 in the
CDD domain with Ser) were transformed into a yeast strain, W303a.
Transformants were grown in glucose-based medium to an OD600 of 1.0,
washed three times with water, and then cultured for 0–24 h in fresh glucose
medium. The optical densities of the media at OD600 were measured (left panel).
The same cultures in the left panel (103 cells) were plated on galactose-based
medium. The number of viable cells was determined after incubating the plates at
301C for 3 days, and the data were normalized to the value of cells cultured in
glucose medium
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Figure 7 AtBAG6 does not interact with AtHSC70. Recombinant GST and
GST-AtBAG3, GST-AtBAG5, and GST-AtBAG6 (560–693) proteins were
expressed in E. coli. In all, 20 mg of crude extract was resolved by 12% SDSPAGE, transferred onto a PVDF membrane, and detected with anti-GST (left
panel). A duplicate membrane was probed with recombinant His-AtHSC70 and
followed by detection with anti-His that is conjugated to horseradish peroxidase
(right panel) to determine if HSP70 can bind to the BAG recombinant proteins
Induction of cell death by AtBAG6 in Arabidopsis
plant
To investigate the function of AtBAG6 in a plant species, we
initially determined AtBAG6 gene expression under various
stress conditions. Total RNA was isolated from stress-treated
Arabidopsis seedlings and Northern blot analysis was
performed using AtBAG6 cDNA as a probe (Figure 8).
Transcription of AtBAG6 was specifically induced by SA,
H2O2, and high temperature, all of which are known to be
involved in plant PCD processes.40–43
To further determine the biological roles of AtBAG6 in
plants, we constructed each plasmid containing CDD or
CDDI575S under the control of the constitutive cauliflower
mosaic virus 35S promoter using the pCAMBIA1302 binary
vector and used these plasmids to transform Arabidopsis.
Transgenic CDD plants showed midget (dwarfism) phenotypes and formed disease-like necrotic lesions on their leaves
(Figure 9b and c). These phenotypes are highly similar to those
of Arabidopsis mutants such as acd1 (accelerated cell death),
cpr1 (constitutive expressor of PR genes), lsd1 (lesion
stimulating disease 1), and agd2 (aberrant growth and death
2), which display a constitutive pathogen response.44–47
Untransformed wild-type, vector control, and CDDI575S transgenic plants grown under identical conditions did not show
these phenotypes (Figure 9). CDD and CDDI157S proteins
were detected immunologically in transgenic lines transformed
with CDD and CDDI157S constructs. These proteins were not
detected in either wild-type or control plants (Figure 9a).
Plant cells undergoing hypersensitive response (HR)-cell
death, which is known to occur via PCD, deposit cell wall
materials including callose and aromatic polymers at their
infected sites.46,48 To determine whether the cell death
(Figure 9c) found in transgenic lines expressing CDD involved
HR-like lesions, plants were stained for callose with aniline
blue and observed under fluorescence microscopy
Figure 8 AtBAG6 transcripts accumulate during the stress response. (a) Total
RNA was prepared from 4-week-old Arabidopsis whole plants treated with
several chemicals (0.1 mM GA4, 2 mM SA, 100 mM JA, 100 mM ABA, 5 mM 2chloroethylphosphonic acid, 100 mM NaCl, 100 mM KCl, or 2 mM H2O2) or
environmental stress (cold; 41C for 6 h, low temperature; 151C for 12 h, high
temperature; 371C for 6 h or drought). A fixed amount of total RNA (20 mg) was
loaded onto each lane. Equal loading for each lane was confirmed by staining
gels with ethidium bromide (lower). An RNA blot was prepared with 32P-labeled
AtBAG6 cDNA. (b) Effect of high temperature on AtBAG6 expression.
Arabidopsis whole plants (4 weeks old) were incubated at 371C for the indicated
times, and total RNA samples were prepared for Northern blot analysis. (c) Effect
of H2O2 on AtBAG6 expression. Arabidopsis whole plants (4 weeks old) were
treated with H2O2 (2 mM) for the indicate time. (d) Effect of SA on the expression
of AtBAG6. Arabidopsis whole plants (4 weeks old) were treated with SA (2 mM)
for the indicated times
(Figure 9d). Whole-mount leaves of CDD transgenic plants
showed prominent abundance of callose, whereas wild-type,
control, and CDDI575S transgenic plants did not (Figure 9d).
Taken together, these results indicate that these necrotic
lesions resemble HR-like lesions.
Discussion
In this report, we present biochemical and functional data in
support of a role for AtBAG6, a BAG domain protein from
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Figure 9 Phenotypes associated with the expression of CDD and CDDI575S in plants. (a) Immunological detection of the 6-His fused CDD and CDDI575S proteins. The
functionalities of these constructs were firstly confirmed in yeast (Figure 5 and data not shown). In all, 40 mg of total proteins from plants shown in (b) were used for
Western blot analysis using anti-His tag antibody conjugated with horseradish peroxidase. The arrow indicates the predicted size of the produced proteins. Col-0, wildtype plant; Control, transgenic plant transformed vector alone; CDD, transgenic plants transformed with CDD (representative lines, #2, #4 were indicated); CDDI575S
transgenic plants transformed with CDDI575S (representative lines, #3, #4 were indicated). (b) Phenotypes associated with expression of CDD in transgenic Arabidopsis.
Photographs were taken 5 weeks after sowing. Lactophenol–trypan blue (c) and aniline blue staining (d) was performed as described in Materials and Methods using the
plants shown in (b)
Arabidopsis, in CaM-mediated cell death. We demonstrate
that (i) oxidative stress, involved in plant PCD processes,40–43
induces the transient expression of AtBAG6, (ii) AtBAG6
physically interacts with AtCaMs, (iii) the IQ motif in the
AtBAG6 is required for Ca2 þ -independent CaM complex
formation, and (iv) the CaM-binding IQ motif and BAG domain
are required for AtBAG6-mediated cell death in both yeast and
plant. In animals, Ca2 þ released into the cytoplasm can
induce mitochondrial permeability transition (PT) pore opening, which induces release of apoptotic activators to the
cytoplasm and consequently activates caspase-mediated
PCD.49,50 Although mechanisms and regulation of plant
PCD are ill-defined, experimental evidences indicate that
Ca2 þ fluxes have pivotal role in the processes. Included are
self-incompatibility of pollen, aleurone differentiation, aerenchyma formation, HR and leaf senescence.51–54 Additional
data confirm a role of CaMBP or channel in pathogen defense.
The Arabidopsis Dnd1 gene, that encodes a cyclic nucleotidedependent calcium channel, is required for the activation of
Cell Death and Differentiation
HR-induced cell death.55 Although the mechanism by which
the CaM–AtBAG6 complex interacts and functions together
with downstream components in planta is currently unclear,
our observations suggest that AtBAG6 may be a specific
component of Ca2 þ /CaM-mediated PCD process in plants.
We screened an Arabidopsis expression library with
CaMHHRP, and identified a novel CaM-binding protein,
AtBAG6. Based on the structural characteristics of known
CaMBDs, the CaM-binding IQ motif was localized to the
middle region of AtBAG6 between Ala572 and Lys591
(Figure 1). This prediction was confirmed by the CaM::HRP
overlay assay (Figures 2 and 3). ‘Complete IQ motifs’
(containing the G and a second basic residue) do not require
Ca2 þ -binding to CaM, whereas ‘incomplete IQ motifs’
(lacking the second basic residue) are Ca2 þ -dependent.56,57
Within the 20 amino-acid stretch (from Ala572 to Lys591) of
AtBAG6 (Figure 1), the conserved residues, the first hydrophobic residue (preceding Q, Ile575), Q (Gln576), and the first
basic residue (Arg580) are present. Furthermore, G (Gly581)
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
91
and the second basic residue (Arg585) are observed. These
features of CaMBD in AtBAG6 are characteristic of the
complete consensus IQ motif.56 Consistent with these finding,
our data confirm that AtBAG6 binds CaM in a Ca2 þ independent manner.
In mammals, some BAG domain proteins regulate diverse
cellular functions by interacting with HSP70/HSC70 and
modulating its activity. For example, the regulation of
HSP70/HSC70 activity by BAG-1 M has been extensively
studied in animals. A number of reports demonstrated that
Glu212, Asp222, Lys238, and Gln245 of BAG-1 are crucial for
these interactions.58,59 However, when we performed either
yeast two-hybrid or in vitro binding assays, we failed to detect
any interactions between AtBAG6 and AtHSC70 (Figure 7
and data not shown). These results suggest that modulation of
HSP70/HSC70 activity is not a universal function of BAG
domain proteins. However, we still cannot rule out the
possibility that AtBAG6 interacts with HSP70/HSC70 in vivo
or that some other factors or specific conditions are required
for this interaction.
Recent studies suggest that the BAG proteins regulate
diverse biochemical events, including receptor signaling,
protein kinase, and transcription factor activity, thereby affect
diverse cellular behaviors ranging from cell division and
differentiation to cell death.60–69 The functional diversity of
BAG domain proteins is paralleled by an abundance of genes
of this family throughout evolution, with homologs identified in
a variety of organisms, including yeast, worm, invertebrates,
amphibians, mammals, and plants.21,59,60,70 Database
searches indicate the presence of eight genes that encode
proteins with the BAG domain in Arabidopsis thaliana.28
Among the proteins encoded by these genes, four (AtBAG5,
AtBAG6, AtBAG7, and AtBAG8) contain a CaMBD (IQ motif)
close to the conserved BAG domain. They might be
functionally redundant since two T-DNA-inserted AtBAG6
mutant lines did not show any significant phenotypic
differences under several stress conditions (heat, cold, salt,
UV, light, or dark) (data not shown). Another possibility is that
they have distinct functions reflected by different spatial and
temporal regulation.71,72 It would be of interest to investigate
the spatial and temporal regulation of AtBAG6 paralogues to
elucidate their specific molecular functions.
Biotic stress-mediated cell death processes, such as HR,
are known to exhibit morphological and biochemical hallmarks
of PCD in mammalian systems. Moreover, since PCD in
plants has been recognized as an integral component of an
adaptive mechanism to stress, scientific opinion on abiotic
stress-mediated cell death has recently shifted.52 Abiotic
stresses, including temperature, salinity, drought, light, ROS,
and ozone, are now often regarded not as toxins, but rather as
elicitors of PCD, when plant cells are chronically exposed to
physiological levels of these stresses.41,73–77 However,
necrosis, which represents an uncontrolled form of cell death,
does occur in plant cells when they are exposed to high levels
of abiotic stress that the plant cannot override with their
tolerance mechanisms. ROS are pivotal mediators of PCD in
plants. ROS are utilized as second messengers in the
execution of cell death during hypersensitivity responses
and ozone-mediated cell death – well-studied plant PCD
phenomenona that are induced by biotic and abiotic stress,
respectively.42 Similarly, AtBAG6-induced cell death in
Arabidopsis and yeast is also mediated by ROS generation
(Figures 4 and 8). Therefore, it would appear that ROS is a
common element and key event of PCD in animals, plants,
and yeast, where it is a common component of a basic,
evolutionarily conserved mechanism.
It has been reported that the expression of BAX (a
mammalian proapoptotic member of the BCL2 family) in
yeast and plants induces apoptosis.78 Although informatics
has failed to identify BAX, BCL2, and BCLXL sequence
homologs in either the Arabidopsis or yeast (Saccharomyces
cerevisiae) genomes, there is evidence that the basic
regulatory mechanisms underlying PCD are conserved in
animal and plant systems.79,80 Experimental evidence of
these conserved mechanisms include caspase-like activities
detected in various plant cell-death model systems, the
demonstration that expression of animal BAX can induce
PCD in plants and yeast, and the identification of the plant
BAX inhibitor-1 (BI-1) that can suppress cell death in both
plants and animals. These strands of evidence indicate the
existence of functional orthologs of BCL2, and its interacting
proteins, in plants. Further identification of components
associated with the CaM-AtBAG6-mediated cellular response
should facilitate elucidation of the mechanism(s) by which
AtBAG6 regulates cell death.
Materials and Methods
Screening of the Arabidopsis cDNA expression
library
A cDNA expression library in a lZAPII vector (Stratagene, La Jolla, CA,
USA) was constructed with RNA from 4-week-old A. thaliana (ecotype
Columbia) plants that were treated with heat shock (371C) for 2 h.
Subsequently, it was screened using horseradish peroxidase (HRP)conjugated Arabidopsis calmodulin-2 (AtCaM2::HRP) as a probe. AtCaM2
was conjugated to maleimide-activated HRP using the EZ-Link maleimideactivated HRP conjugation kit (Pierce, Rockford, IL, USA), as described in
a previous report.81 Approximately 5 105 pfu cells were plated per 15 cm
LB plate, using E. coli XL1-blue MRF (Stratagene, La Jolla, CA, USA) as
the host strain. Plates were incubated at 421C until plaques appeared, and
overlaid with nitrocellulose filters previously soaked in 10 mM IPTG.
Incubation was continued at 371C for 6–8 h, and plates were cooled to
41C. Filters were removed, and rinsed twice in a large volume of TBS-T
(Tris-based saline containing 0.1% (v/v) Tween-20). Next, filters were
blocked by incubation in 7% (w/v) nonfat dry milk/TBS-T overnight.
Blocked filters were washed three times with TBS-T for 5 min and
equilibrated in overlay buffer (50 mM imidazole-HCl (pH 7.5), 150 mM
NaCl) for 1 h. Membranes were blocked secondly by incubating filters in
overlay buffer containing 9% (v/v) gelatin (Sigma-Aldrich, St. Louis, MO,
USA), 0.5% (v/v) Tween-20, and 5 mM EGTA for 3.5 h. AtCaM2::HRP was
added to gelatin-containing buffer at a final concentration of 0.2 mg/ml, and
filters were incubated for 1 h. The final washing was performed in three
steps, with each step consisting of five repeats of a 5-min wash: firstly in
TBS-T/50 mM imidazole-HCl (pH 7.5) and 5 mM EGTA; secondly in
20 mM Tris-HCl (pH 7.5), 0.5% Tween-20, 50 mM imidazole-HCl, 0.5 M
KCl, and 5 mM EGTA; and thirdly in 20 mM Tris-HCl (pH 7.5), 0.1%
Tween-20, and 1 mM MgCl2. Bound AtCaM2::HRP was visualized using
an enhanced chemiluminescence (ECL) detection kit (Amersham
Pharmacia Biotech, Uppsala, Sweden). A total of 5 105 recombinants
Cell Death and Differentiation
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
92
were screened, and 11 positive clones isolated after three rounds of
screening. cDNA inserts were recovered by in vivo excision with helper
phage (ExAssist, Stratagene, La Jolla, CA, USA). To confirm binding to
AtCaM2, we expressed positive clones as b-galactosidase fusion proteins
in E. coli. Clones were examined for CaM-binding by AtCaM2::HRP
overlay assay, as described above. In brief, we transformed positive
clones into E. coli XL1-blue MRF and induced the expression of
b-galactosidase fusion proteins by treatment with 0.5 mM IPTG. IPTGinduced E. coli crude proteins (20 mg) were separated on a 10% SDSpolyacrylamide gel, and transferred to an Immobilon-MP membrane
(PVDF, Millipore, Bedford, MA, USA). The membrane was rinsed in TBST, blocked by incubation in 7% (w/v) nonfat dry milk/TBS-T overnight, and
processed as described above. For determination of Ca2 þ -dependent
binding of CaM, 1 mM CaCl2 was substituted for 5 mM EGTA in all overlay
buffers. The cDNA sequences of the resulting positive clones were
determined from both strands by dideoxynucleotide chain termination
using an automatic DNA sequencer (ABI 373A, Applied Biosystems,
Foster City, CA, USA).
Yeast two-hybrid assays
The full-length coding region of AtBAG6 was cloned into the pGBT9 vector
(encoding the TRP1 gene, Clontech, Palo Alto, CA, USA) containing the
GAL4 DNA-binding domain (BD). An Arabidopsis cDNA library was
constructed into the pGAD424 vector (including the LEU2 gene, Clontech,
Palo Alto, CA, USA) containing the GAL4 activation domain (AD). pTD1-1
and pVA3-1 (Clontech, Palo Alto, CA, USA) encoding the interacting
proteins, tumor suppressor p53 and simian virus 40 (SV40) large Tantigen fused to BD and AD, respectively, were used as positive
controls.82 The pGAD424 vector containing the Arabidopsis cDNA library
was transformed into the yeast reporter strain pJ69-4A (MATa trp1-90
leu2-3,112 ura3-52 his3-200 gal4 Dgal80 DLYS2::GAL1-HIS3 GAL2ADE2 met2::GAL7-lacZ) harboring pGBT9-AtBAG6.83 Interactions between the encoded fusion proteins were investigated by cotransforming
appropriate plasmids into the yeast strain pJ69-4. Transformed yeast cells
bearing both the plasmids were selected by plating on SD medium (0.67%
nitrogen base without amino acids, amino acids and nucleotide bases)
lacking tryptophan and leucine (SD-WL), and grown at 301C for 4 days.
We tested the interactions of proteins encoded by recombinant pGBT9/
pGAD424 by growing cells in SD medium lacking tryptophan, leucine, and
adenine (SD-WLA). Adenine-positive colonies were further tested for bgalactosidase (LacZ) activation, according to the manufacturer’s protocol
(Clontech, Palo Alto, CA, USA).
Construction of deletion mutants of AtBAG6 cDNA
and site-directed mutagenesis
For mapping of the CaM-binding domain, several fragment constructs
were generated in a pGEX-5X-2 vector (Amersham Pharmacia Biotech,
Uppsala, Sweden). To observe the phenotypes of yeast cells overexpressing several genes, we used pYES2-GST fusion vector, which had
been derived from pYES2 vector (Invitrogen, Carlsbad, CA, USA). A KpnI–
BamHI fragment has been amplified by polymerase chain reaction (PCR)
with pGEX-5X-2 vector as template and primers designed to amplify the
vector residues 258–940, which encode the 26 kDa glutathione Stransferase (GST). The fragment was inserted into a pYES2 vector
digested with KpnI and BamHI to produce the pYES2-GST fusion vector.
The full-length AtBAG6 cDNA clone was amplified by PCR with a
forward (50 ) primer containing a BglII site (50 -AGATCTTAAT
GATGCCTGTGTACATGGA-30 ) and a reverse (30 ) primer containing a
XhoI site (50 -CCTCGAGGTCATAATACGGCATCGGT-30 ). The PCR
Cell Death and Differentiation
product was cloned into the pGEM-T vector (Promega, Madison, WI,
USA) and sequenced to verify the correct construct. The construct was
digested with BglII and XhoI and subcloned into the pGEX-5X-2
expression vector digested with BamHI and XhoI. This full-length GSTfusion construct was designated D0 (encompassing amino acids 1–1043).
To analyze the regions to bind CaM or to induce cell death in yeast, serial
fragment constructs additionally generated by PCR using the following
forward (F) and reverse (R) primer sequences: for D1 (amino acids 1–
579): F, containing a BglII site (50 -AGATCTTAATGATGCCTGTGTA
CATGGA-30 ) and R, containing a XhoI site (50 -CATCTCGAGGCTACA
TAGATTG-30 ); for D2 (amino acids 1–592): F, containing a BglII site (50 AGATCTTAATGATGCCTGTGTACATGGA-30 ) and R, containing a XhoI
site (50 -CTCGAGCAATTACTTAATTGG-30 ); for D3 (amino acids 560–
1043): F, containing a BamHI site (50 -GGAGAATGGATCCAGCCTGC-30 )
and R, containing a XhoI site (50 -CCTCGAGGTCATAATACGG
CATCGGT-30 ); for D4 (amino acids 584–1043): F, containing a BamHI
site (50 -GTACCGTGGATCCACGTGAGAAG-30 ) and R, containing a XhoI
site (50 -CCTCGAGGTCATAATACGGCATCGGT-30 ); for CDD (amino
acids 560–693): F, containing a BamHI site (50 -GGAGAATGGATC
CAGCCTGC-30 ) and R, containing a XhoI site (50 - CCTCGAGAGGCT
GAGATTTAATTTCCAC-30 ). The amplified products were cloned into the
pGEM-T vector, and subcloned into the pGEX-5X-2 expression vector
digested with BamHI and XhoI for E. coli or pYES2-GST fusion vector
digested with BamHI and XhoI for yeast.
To identify the critical residues in the interactions between CaM and
AtBAG6, we introduced several point mutations into the IQ motif of CDD
(CaMBD). Substitution of single amino acids was performed using the
QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA,
USA). The following F and R primers were employed: for I575V: F, 50 GCTAGAATTGTCCAATCTATG-30 and R, 50 -CATAGATTGGACAATTC
TAGC-30 ; for I575S: F, 50 -GCTAGAATTAGCCAATCTATG-30 and R, 50 CATAGATTGGCTAATTCTAGC-30 ; for I575N: F, 50 -GCTAGAATTAA
CCAATCTATG-30 and R, 50 -CATAGATTGGTTAATTCTAGC-30 .
Production of recombinant proteins in E. coli and
the CaM-binding assay
All clones were introduced into E. coli BL21(DE3)pLysS. Production of the
6-His tag fusion or GST fusion proteins was induced by the application of
1 mM IPTG for 5 h at 251C. Cells were harvested, resuspended in lysis
buffer (50 mM Tris-HCl (pH 7.5), 2 mM PMSF, 1 mM DTT, 100 mg/ml
lysozyme), and incubated on ice for 20 min. The mixture was sonicated for
1 min at 50% pulse and centrifuged at 6000 g for 10 min to remove cell
debris. The supernatant (containing the E. coli crude protein) was used for
Western blotting and AtCaM2::HRP gel overlay assay. E. coli crude
protein (20 mg) was separated on 10% SDS-polyacrylamide gels, and
transferred to Immobilon-P membranes (PVDF, Millipore, Bedford, MA,
USA). Expressed 6-His tag fusion or GST fusion proteins were detected
with the appropriate specific antisera. For far-Western blotting, the 6-His
tag was detected by His-probe::HRP (Santa Cruz Biotechnology, CA,
USA).84 To determine the CaM-binding abilities of recombinant proteins, a
duplicate blot was probed with an AtCaM2::HRP conjugate in the
presence of 5 mM EGTA or 1 mM CaCl2. The AtCaM2::HRP overlay assay
was performed as described above. Bound CaM was visualized using an
ECL detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
Yeast strains, spot assay, and viability assay
Plasmids containing full-length AtBAG6 or a serial fragment were cloned
into the pYES2-GST fusion vector, as described above. Additionally BAX,
AtBAG1, and AtBAG8 genes were cloned by PCR and subcloned into
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
93
pYES2-GST. The plasmids were transformed into the wild-type S.
cerevisiae strain, W303-1a (MATa ura3-1, leu2-3, 112 his3-11, 15 ade2-1
trp1-1 can1-110).85 For spot assays, strains were pregrown in SD medium
lacking uracil in the presence of 2% glucose as the carbon source (SD-U/
Glu) at 301C to a cell density of about 0.5 106/cm3. After washing three
times, aliquots (10 ml) of 10-fold serial dilutions were spotted on plates of
SD medium lacking uracil, in the presence of 2% glucose (SD-U/Glu) or
2% galactose (SD-U/Gal) as the carbon source. Plates were incubated at
301C, and examined for surviving cells after 4 days.
To determine the viabilities of strains, cells were pregrown in SD
medium lacking uracil, and containing 2% glucose (SD-U/Glu) as
described above, and pelleted by centrifugation (1000 g) for 10 min.
After washing three times, cells were resuspended in 10 ml SD medium
lacking uracil in the presence of 2% galactose (SD-U/Gal) to an optical
density at 600 nm (OD600) of 1.3. After culturing for various times (0–24
days), the optical densities of media at 600 nm (OD600) were measured
after four-fold dilution. Aliquots of cells were plated on SD medium lacking
uracil in the presence 2% glucose (SD-U/Glu). Plates were incubated at
301C for 4 days, and the numbers of colonies were counted.
Microscopic examination
For microscopic examination, strains were pregrown in SD medium
lacking uracil and containing 2% glucose (SD-U/Glu), as described above.
After washing three times, cells were cultured in SD medium lacking uracil
in the presence of 2% galactose (SD-U/Gal) for 12 h. TdT-mediated dUTP
nick end labeling (TUNEL) tests were performed with the In Situ Cell Death
Detection Kit, Fluorescein (Roche Applied Science, Indianapolis, IN, USA)
as described by Madeo et al.38 Phosphatidylserine exposure was detected
by an FITC-coupled annexin V reaction with the ApoAlert Annexin V
Apoptosis kit (Clontech, Palo Alto, CA, USA), essentially as described by
Ludovico et al.34 To visualize nuclei, cells were incubated with 1 mg/ml
DAPI in HEPES buffer (10 mM HEPES/NaOH buffer pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2) for 20 min, washed three times with HEPES buffer,
and examined under a fluorescence microscope. To determine ROS
generation, cells were rewashed in water, resuspended in Tris buffer
(50 mM Tris-HCl, pH 7.5), incubated for 2 h at room temperature with 5 mg/
ml dihydrorhodamine123 (Molecular Probes, Eugene, OR, USA), and
examined under a fluorescence microscope.85
Northern blot analysis
Whole Arabidopsis plants (4 weeks old) were treated with several
chemicals (0.1 mM GA4, 2 mM SA, 100 mM JA, 100 mM ABA, 5 mM 2chloroethylphosphonic acid, 100 mM NaCl, 100 mM KCl, 2 mM H2O2) and
environmental stress conditions (cold; 41C for 6 h, low temperature; 151C
for 12 h, high temperature; 371C for 6 h and drought).
Total RNA was isolated by phenol/chloroform extraction, followed by
lithium chloride precipitation.86 RNA (20 mg) was denatured, separated by
electrophoresis on a 1.2% (w/v) agarose-formaldehyde gel, and
transferred to a nylon membrane (GeneScreen Plus, PerkinElmer Life
and Analytical Sciences, Boston, MA, USA). Membranes were incubated
with 32P-labeled full-length AtBAG6 cDNA at 651C overnight, and washed
under high stringency conditions according to the method of Church and
Gilbert.87
Construction of transgenic plants and tests for
PCD
For experiments with Arabidopsis, A. thaliana (ecotype Columbia) was
used. The plants were grown on a solid medium containing basic MS salts
(Duchefa Biochemical Co., Netherlands), 3% sucrose, and 0.8% agar. To
generate overexpressing transgenic lines, we cloned full-length AtBAG6,
CDD or CDDI575S which had been N-terminally fused to GST coding region
downstream of the cauliflower mosaic virus 35S promoter, in the sense
orientation into the pCAMBIA1302 binary vector. For control lines, only an
empty vector construct was used. These plasmid constructs were first
transformed into Agrobacterium tumefaciens GV3101 and then
subsequently into Arabidopsis wild-type (Col-0) backgrounds. Kanamycin
was used for bacterial selection and hygromycin B for plant selection.
Plants of the T0 generation were grown to maturity and T1 seeds were
harvested. T1 progeny of transgenic plants expressing high levels of
GST-CDD or GST-CDDI575S were used for all of the experiments. For the
PCD experiments, 2-week-old seedlings on solid agar plates containing
30 mg/ml hygromycin B were transferred to soil and maintained at
221C under long-day conditions (16 h light/8 h dark). After growth for 4
weeks on soil, the fourth leaves were detached and were tested for PCD.
Cell death was detected by lactophenol-trypan blue staining as described
by Koch and Slusarenko.88 For aniline blue staining of callose,
plant samples were boiled in ethanol/lactophenol (2:1 (v/v)) for 20 min.
Samples were then rinsed with water to remove the lactophenol and
stained for 1 h with aniline blue (0.01% aniline blue powder in 150 mM
K2PO4, pH 9.5). Before samples were mounted, they were equilibrated in
50% glycerol. Aniline blue staining was visualized by fluorescence
microscopy.89
Acknowledgements
This research was partially supported by grants (PF0330401-00) from the
Plant Diversity Research Center of the 21st Century Frontier Research
Program, MOST, the Environmental Biotechnology Core Research Center
(R15-2003-012-01002-0) from KOSEF/MOST, KRIBB Research initiative
program, and Bigreen 21 program, Rural Development Administration,
Korea. CH Kang, WY Jung, JC Jeong, DW Baek were supported by
scholarships from the Brain Korea 21 program, Ministry of Education,
Korea.
References
1. McAinsh MR and Hetherington AM (1998) Encoding specificity in Ca2+
signalling systems. Trends Plant Sci. 3: 32–36
2. Trewavas AJ and Malho RC (1998) Ca2+ signalling in plant cells: the big
network!. Curr. Opin. Plant Biol. 1: 428–433
3. Dolmetsch RE, Lewis RS, Goodnow CC and Healy JI (1997) Differential
activation of transcription factors induced by Ca2+ response amplitude and
duration. Nature 386: 855–858
4. Snedden WA and Fromm H (2001) Calmodulin as a versatile calcium signal
transducer in plants. New Phytol. 151: 35–66
5. Hoeflich KP and Ikura M (2002) Calmodulin in action: diversity in target
recognition and activation mechanisms. Cell. Cell 108: 739–742
6. Yokouchi T, Izumi Y, Matsufuji T, Jinbo Y and Yoshino H (2003) Unfolding
intermediate of a multidomain protein, calmodulin, in urea as revealed by smallangle X-ray scattering. FEBS Lett. 551: 119–122
7. Alexander KA, Wakim BT, Doyle GS, Walsh KA and Storm DR (1988)
Identification and characterization of the calmodulin-binding domain of
neuromodulin, a neurospecific calmodulin-binding protein. J. Biol. Chem.
263: 7544–7549
8. Baudier J, Deloulme JC, Van Dorsselaer A, Black D and Matthes HW (1991)
Purification and characterization of a brain-specific protein kinase C substrate,
neurogranin (p17). Identification of a consensus amino acid sequence between
neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase
Cell Death and Differentiation
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
94
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
C phosphorylation site and the calmodulin-binding domain. J. Biol. Chem. 266:
229–237
Mitchell EJ, Karn J, Brown DM, Newman A, Jakes R and Kendrick-Jones J
(1989) Regulatory and essential light-chain-binding sites in myosin heavy chain
subfragment-1 mapped by site-directed mutagenesis. J. Mol. Biol. 208: 199–
205
McNally EM, Bravo-Zehnder MM and Leinwand LA (1991) Identification of
sequences necessary for the association of cardiac myosin subunits. J. Cell.
Biol. 113: 585–590
Kiegle E, Moore CA, Haseloff J, Tester MA and Knight MR (2000) Cell-typespecific calcium responses to drought, salt and cold in the Arabidopsis root.
Plant J. 23: 267–278
Knight H (2000) Calcium signaling during abiotic stress in plants. Int. Rev.
Cytol. 195: 269–324
Zielinski RE (1998) Calmodulin and calmodulin-binding proteins in plants.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 697–725
Reddy VS, Ali GS and Reddy AS (2002) Genes encoding calmodulin-binding
proteins in the Arabidopsis genome. J. Biol. Chem. 277: 9840–9852
Yang T and Poovaiah BW (2003) Calcium/calmodulin-mediated signal network
in plants. Trends Plant Sci. 8: 505–512
Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA and Reed JC
(1995) Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein
with anti-cell death activity. Cell 80: 279–284
Takayama S, Xie Z and Reed JC (1999) An evolutionarily conserved family of
Hsp70/Hsc70 molecular chaperone regulators. J. Biol. Chem. 274: 781–786
Doong H, Vrailas A and Kohn EC (2002) What’s in the ‘BAG’ ? – A functional
domain analysis of the BAG-family proteins. Cancer Lett. 188: 25–32
Hung WJ, Roberson RS, Taft J and Wu DY (2003) Human BAG-1 proteins bind
to the cellular stress response protein GADD34 and interfere with GADD34
functions. Mol. Cell. Biol. 23: 3477–3486
Yoo JH, Cheong MS, Park CY, Moon BC, Kim MC, Kang YH, Park HC, Choi
MS, Lee JH, Jung WY, Yoon HW, Chung WS, Lim CO, Lee SY and Cho MJ
(2004) Regulation of the dual specificity protein phosphatase, DsPTP1, through
interactions with calmodulin. J. Biol. Chem. 279: 848–858
Kim MC, Panstruga R, Elliott C, Muller J, Devoto A, Yoon HW, Park HC, Cho
MJ and Schulze-Lefert P (2002) Calmodulin interacts with MLO protein to
regulate defence against mildew in barley. Nature 416: 447–451
Kim MC, Lee SH, Kim JK, Chun HJ, Choi MS, Chung WS, Moon BC, Kang CH,
Park CY, Yoo JH, Kang YH, Koo SC, Koo YD, Jung JC, Kim ST, Schulze-Lefert
P, Lee SY and Cho MJ (2002) Mlo, a modulator of plant defense and cell death,
is a novel calmodulin-binding protein. Isolation and characterization of a rice
Mlo homologue. J. Biol. Chem. 277: 19304–19314
Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP, Ranjeva R and
Ranty B (2004) A novel calmodulin-binding protein functions as a negative
regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings. Plant J.
38: 410–420
Juqiang Y, Cixin H and Hong Z (2003) The BAG-family proteins in Arabidopsis
thaliana. Plant Sci. 165: 1–7
Jurado LA, Chockalingam PS and Jarrett HW (1999) Apocalmodulin. Physiol.
Rev. 79: 661–682
Rhoads AR and Friedberg F (1997) Sequence motifs for calmodulin
recognition. FASEB J. 11: 331–340
Kawasaki H, Nakayama S and Kretsinger RH (1998) Classification and
evolution of EF-hand proteins. BioMetals. 11: 277–295
Cheney RE and Mooseker MS (1992) Unconventional myosins. Curr. Opin.
Cell Biol. 4: 27–35
Poetsch A, Molday LL and Molday RS (2001) The cGMP-gated channel
and related glutamic acid-rich proteins interact with peripherin-2 at
the rim region of rod photoreceptor disc membranes. J. Biol. Chem. 276:
48009–48016
Korschen HG, Beyermann M, Muller F, Heck M, Vantler M, Koch KW, Kellner
R, Wolfrum U, Bode C, Hofmann KP and Kaupp UB (1999) Interaction of
glutamic-acid-rich proteins with the cGMP signalling pathway in rod
photoreceptors. Nature 400: 761–766
Liu J, Seul U and Thompson R (1997) Cloning and characterization of a pollenspecific cDNA encoding a glutamic-acid-rich protein (GARP) from potato
Solanum berthaultii. Plant Mol. Biol. 33: 291–300
Gross A, McDonnell JM and Korsmeyer SJ (1999) BCL-2 family members and
the mitochondria in apoptosis. Genes Dev. 13: 1899–1911
Cell Death and Differentiation
33. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC,
LaFace DM and Green DR (1995) Early redistribution of plasma membrane
phosphatidylserine is a general feature of apoptosis regardless of the initiating
stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182: 1545–
1556
34. Ludovico P, Sousa MJ, Silva MT, Leao C and Corte-Real M (2001)
Saccharomyces cerevisiae commits to a programmed cell death process in
response to acetic acid. Microbiology 147: 2409–2415
35. Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B and von Arnold
S (2000) Two waves of programmed cell death occur during formation and
development of somatic embryos in the gymnosperm, Norway spruce. J. Cell
Sci. 113: 4399–4411
36. Fath A, Bethke P, Beligni V and Jones R (2002) Active oxygen and cell death in
cereal aleurone cells. J. Exp. Bot. 53: 1273–1282
37. Jabs T (1999) Reactive oxygen intermediates as mediators of programmed cell
death in plants and animals. Biochem. Pharmacol. 57: 231–245
38. Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH and Frohlich KU
(1999) Oxygen stress: a regulator of apoptosis in yeast. J. Cell. Biol. 145: 757–
767
39. Schulz JB, Weller M and Klockgether T (1996) Potassium deprivation-induced
apoptosis of cerebellar granule neurons: a sequential requirement for new
mRNA and protein synthesis, ICE-like protease activity, and reactive oxygen
species. J. Neurosci. 16: 4696–4706
40. Lamb C and Dixon RA (1997) The oxidative burst in plant disease resistance.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 251–275
41. Banzet N, Richaud C, Deveaux Y, Kazmaier M, Gagnon J and Triantaphylides
C (1998) Accumulation of small heat shock proteins, including mitochondrial
HSP22, induced by oxidative stress and adaptive response in tomato cells.
Plant J. 13: 519–527
42. Rao MV and Davis KR (1999) Ozone-induced cell death occurs via two distinct
mechanisms in Arabidopsis: the role of salicylic acid. Plant J. 17: 603–614
43. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends
Plant Sci. 7: 405–410
44. Greenberg JT and Ausubel FM (1993) Arabidopsis mutants compromised for
the control of cellular damage during pathogenesis and aging. Plant J. 4: 327–
341
45. Bowling SA, Guo A, Cao H, Gordon AS, Klessig DF and Dong X (1994) A
mutation in Arabidopsis that leads to constitutive expression of systemic
acquired resistance. Plant Cell. 6: 1845–1857
46. Dietrich RA, Delaney TP, Uknes SJ, Ward ER, Ryals JA and Dangl JL (1994)
Arabidopsis mutants simulating disease resistance response. Cell 77: 565–577
47. Rate DN and Greenberg JT (2001) The Arabidopsis aberrant growth and
death2 mutant shows resistance to Pseudomonas syringae and reveals a role
for NPR1 in suppressing hypersensitive cell death. Plant J. 27: 203–211
48. Holt III BF, Mackey D and Dangl JL (2000) Recognition of pathogens by plants.
Curr Biol. 10: 5–7
49. Scorrano L and Korsmeyer SJ (2003) Mechanisms of cytochrome c release by
proapoptotic BCL-2 family members. Biochem. Biophys. Res. Commun. 304:
437–444
50. Demaurex N and Distelhorst C (2003) Cell biology. Apoptosis – the calcium
connection. Science 300: 65–67
51. Thomas SG and Franklin-Tong VE (2004) Self-incompatibility triggers
programmed cell death in Papaver pollen. Nature 429: 305–309
52. Dangl JL, Dietrich RA and Thomas H (2000) Senescence and programmed cell
death In Biochemistry and Molecular Biology of Plants, Buchanan B, Gruisem
W and Jones R (eds) MD: American Society Plant Physiology pp. 1044–1100
53. Levine A, Pennell RI, Alvarez ME, Palmer R and Lamb C (1996) Calciummediated apoptosis in a plant hypersensitive disease resistance response. Curr
Biol. 6: 427–437
54. Jongebloed U, Szederkenyi J, Hartig K, Schobert C and Komor E (2004)
Sequence of morphological and physiological events during natural ageing and
senescence of a castor bean leaf: sieve tube occlusion and carbohydrate backup precede chlorophyll degradation. Physiol Plant. 120: 338–346
55. Clough SJ, Fengler KA, Yu IC, Lippok B, Smith Jr RK and Bent AF (2000) The
Arabidopsis dnd1 ‘defense, no death’ gene encodes a mutated cyclic
nucleotide-gated ion channel. Proc. Natl. Acad. Sci. USA 97: 9323–9328
56. Houdusse A and Cohen C (1995) Target sequence recognition by the
calmodulin superfamily: implications from light chain binding to the regulatory
domain of scallop myosin. Proc. Natl. Acad. Sci. USA 92: 10644–10647
A CaM-binding BAG domain protein in Arabidopsis
CH Kang et al
95
57. Munshi HG, Burks DJ, Joyal JL, White MF and Sacks DB (1996) Ca2+ regulates
calmodulin binding to IQ motifs in IRS-1. Biochemistry 35: 15883–15889
58. Sondermann H, Ho AK, Listenberger LL, Siegers K, Moarefi I, Wente SR, Hartl
FU and Young JC (2002) Prediction of novel Bag-1 homologs based on
structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in
Saccharomyces cerevisiae. J. Biol. Chem. 277: 33220–33227
59. Sondermann H, Scheufler C, Schneider C, Hohfeld J, Hartl FU and Moarefi I
(2001) Structure of a Bag/Hsc70 complex: convergent functional evolution of
Hsp70 nucleotide exchange factors. Science 291: 1553–1557
60. Froesch BA, Takayama S and Reed JC (1998) BAG-1L protein enhances
androgen receptor function. J. Biol. Chem. 273: 11660–11666
61. Schneikert J, Hübner S, Martin E and Cato AC (1999) A nuclear action of the
eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor
activity. J. Cell Biol. 146: 929–940
62. Wang HG, Takayama S, Rapp UR and Reed JC (1996) Bcl-2 interacting
protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl. Acad. Sci.
USA 93: 7063–7068
63. Bardelli A, Longati P, Albero D, Goruppi S, Schneider C, Ponzetto C and
Comoglio PM (1996) HGF receptor associates with the anti-apoptotic protein
BAG-1 and prevents cell death. EMBO J. 15: 6205–6212
64. Matsuzawa S, Takayama S, Froesch BA, Zapata JM and Reed JC (1998) p53inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell
growth: suppression by BAG-1. EMBO J. 17: 2736–2747
65. Kullmann M, Schneikert J, Moll J, Heck S, Zeiner M, Gehring U and Cato AC
(1998) RAP46 is a negative regulator of glucocorticoid receptor action and
hormone-induced apoptosis. J. Biol. Chem. 273: 14620–14625
66. Liu R, Takayama S, Zheng Y, Froesch B, Chen G, Zhang X, Reed JC and
Zhang X (1998) Interaction of BAG-1 with retinoic acid receptor and its inhibition
of retinoic acid-induced apoptosis in cancer cells. J. Biol. Chem. 273: 16985–
16992
67. Naishiro Y, Adachi M, Okuda H, Yawata A, Mitaka T, Takayama S, Reed JC,
Hinoda Y and Imai K (1999) BAG-1 accelerates cell motility of human gastric
cancer cells. Oncogene 18: 3244–3251
68. Zeiner M, Niyaz Y and Gehring U (1999) The hsp70-associating protein Hap46
binds to DNA and stimulates transcription. Proc. Natl. Acad. Sci. USA 96:
10194–10199
69. Tschopp J, Martinon F and Hofmann K (1999) Apoptosis: silencing the death
receptors. Curr. Biol. 9: 381–384
70. Emanuelsson O, Nielsen H, Brunak S and von Heijne G (2000) Predicting
subcellular localization of proteins based on their N-terminal amino acid
sequence. J. Mol. Biol. 300: 1005–1016
71. Bannai H, Tamada Y, Maruyama O, Nakai K and Miyano S (2002) Extensive
feature detection of N-terminal protein sorting signals. Bioinformatics 18: 298–
305
72. Suzuki M, Youle RJ and Tjandra N (2000) Structure of Bax: coregulation of
dimer formation and intracellular localization. Cell 103: 645–654
73. Huh GH, Damsz B, Matsumoto TK, Reddy MP, Rus AM, Ibeas JI, Narasimhan
ML, Bressan RA and Hasegawa PM (2002) Salt causes ion disequilibriuminduced programmed cell death in yeast and plants. Plant J. 29: 649–659
74. Vacca RA, de Pinto MC, Valenti D, Passarella S, Marra E and De Gara L (2004)
Production of reactive oxygen species, alteration of cytosolic ascorbate
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
peroxidase, and impairment of mitochondrial metabolism are early events in
heat shock-induced programmed cell death in tobacco Bright-Yellow 2 cells.
Plant Physiol. 134: 1100–1112
Tiwari BS, Belenghi B and Levine A (2002) Oxidative stress increased
respiration and generation of reactive oxygen species, resulting in ATP
depletion, opening of mitochondrial permeability transition, and programmed
cell death. Plant Physiol. 128: 1271–1281
Nibbe M, Hilpert B, Wasternack C, Miersch O and Apel K (2002) Cell death and
salicylate- and jasmonate-dependent stress responses in Arabidopsis are
controlled by single cet genes. Planta 216: 120–128
Xiao S, Brown S, Patrick E, Brearley C and Turner JG (2003) Enhanced
transcription of the Arabidopsis disease resistance genes RPW8.1 and
RPW8.2 via a salicylic acid-dependent amplification circuit is required for
hypersensitive cell death. Plant Cell. 15: 33–45
Kawai-Yamada M, Jin L, Yoshinaga K, Hirata A and Uchimiya H (2001)
Mammalian Bax-induced plant cell death can be down-regulated by
overexpression of Arabidopsis Bax Inhibitor-1 (AtBI-1). Proc. Natl. Acad. Sci.
USA 98: 12295–12300
Elbaz M, Avni A and Weil M (2002) Constitutive caspase-like machinery
executes programmed cell death in plant cells. Cell Death Differ. 9: 726–733
Lacomme C and Santa Cruz S (1999) Bax-induced cell death in tobacco is
similar to the hypersensitive response. Proc. Natl. Acad. Sci. USA 96: 7956–
7961
Lee SH, Kim MC, Heo WD, Kim JC, Chung WS, Park CY, Park HC,
Cheong YH, Kim CY, Lee KJ, Bahk JD, Lee SY and Cho MJ (1999) Competitive
binding of calmodulin isoforms to calmodulin-binding proteins: implication for
the function of calmodulin isoforms in plants. Biochim. Biophys. Acta
1433: 56–67
Iwabuchi K, Li B, Bartel P and Fields S (1993) Use of the two-hybrid system to
identify the domain of p53 involved in oligomerization. Oncogene 8: 1693–1696
James P, Halladay J and Craig EA (1996) Genomic libraries and a host strain
designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425–
1436
Iftner T, Elbel M, Schopp B, Hiller T, Loizou JI, Caldecott KW and Stubenrauch
F (2002) Interference of papillomavirus E6 protein with single-strand break
repair by interaction with XRCC1. EMBO J. 21: 4741–4748
Moon H, Baek D, Lee B, Prasad DT, Lee SY, Cho MJ, Lim CO, Choi MS, Bahk
J, Kim MO, Hong JC and Yun DJ (2002) Soybean ascorbate peroxidase
suppresses Bax-induced apoptosis in yeast by inhibiting oxygen radical
generation. Biochem. Biophys. Res. Commun. 290: 457–462
Lagrimini LM, Burkhart W, Moyer M and Rothstein S (1987) Molecular cloning
of complementary DNA encoding the lignin-forming peroxidase from tobacco:
molecular analysis and tissue-specific expression. Proc. Natl. Acad. Sci. USA
84: 7542–7546
Church GM and Gilbert W (1984) Genomic sequencing. Proc. Natl. Acad. Sci.
USA 81: 1991–1995
Koch E and Slusarenko A (1990) Arabidopsis is susceptible to infection by a
downy mildew fungus. Plant Cell 2: 437–445
Stone JM, Heard JE, Asai T and Ausubel FM (2000) Simulation of fungalmediated cell death by fumonisin B1 and selection of fumonisin B1-resistant
(fbr) Arabidopsis mutants. Plant Cell 12: 1811–1822
Supplementary Information accompanies the paper on Cell Death Differentiation website (http://www.nature.com/cdd).
Cell Death and Differentiation